Recombinant genes for producing proteins in plants comprise in sequence a promoter which functions in plants, a structural gene encoding the target protein, and a non-translated region that functions in plants to cause the addition of polyadenylated nucleotides to the RNA sequence. Much scientific effort has been directed to improve these recombinant plant genes to express larger amounts of the target protein.
One advantage of higher levels of expression is that fewer numbers of transgenic plants need to be produced and screened to recover plants producing sufficient quantities of the target protein to be agronomically significant. High level expression leads to plants exhibiting commercially important phenotypical properties.
Improved recombinant plant genes have been found by use of more potent promoters, such as promoters from plant viruses. Further improvement in expression has been obtained in gene constructs by placing enhancer sequences 5' the promoter. Still further improvement has been achieved, especially in monocot plants by gene constructs having introns in the non-translated leader positioned between the promoter and the structural gene coding sequence. For example, Callis et al. (1987) Genes and Development, Vol. 1, pp. 1183-1200, reported that the presence of alcohol dehydrogenase-1 (Adh-1) introns or Bronze-1 introns resulted in higher levels of expression. Dietrich et al. (1987) J. Cell Biol., 105, p. 67, reported that the 5' untranslated leader length was important for gene expression in protoplast. Mascarenkas et al. (1990) Plant Mol. Biol., Vol. 15, pp. 913-920, reported a 12-fold and 20-fold enhancement of CAT expression by use of the Adh-1 intron. Vasil et al. (1989) Plant Physiol., 91, pp. 1575-1579, reported that the Shrunken-1 (Sh-1) intron gave about 10 times higher expression than constructs containing the Adh-1 intron. Silva et al. (1987) J. Cell Biol., 105, p. 245, reported a study of the effect of the untranslated region of the 18 Kd heat shock protein (HSP18) gene on expression of CAT. Semrau et al. (1989) J. Cell Biol., 109, p. 39A, and Mettler et al., N.A.T.O. Advanced Studies Institute on Molecular Biology, Elmer, Bavaria (May 1990) reported that the 140 bp intron of the 82 Kd heat shock protein (HSP82) enhanced expression in maize protoplasts.
The search for even more improved recombinant plant genes continues for the reasons discussed above.